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CDP-2 3-Di-O-Geranylgeranyl-sn-Glycerol:L-Serine O-Archaetidyltransferase(Archaetidylserine Synthase) in the Methanogenic Archaeon Methanothermobacte
http://www.100md.com 《细菌学杂志》2003年第4期
     Department of Chemistry, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan6uj, http://www.100md.com

    Received 8 August 2002/ Accepted 18 November 20026uj, http://www.100md.com

    ABSTRACT6uj, http://www.100md.com

    CDP-2,3-di-O-geranylgeranyl-sn-glycerol:L-serine O-archaetidyltransferase (archaetidylserine synthase) activity in cell extracts of Methanothermobacter thermautotrophicus cells was characterized. The enzyme catalyzed the formation of unsaturated archaetidylserine from CDP-unsaturated archaeol and L-serine. The identity of the reaction products was confirmed by thin-layer chromatography, fast atom bombardment-mass spectrum analysis, and chemical degradation. The enzyme showed maximal activity in the presence of 10 mM Mn2+ and 1% Triton X-100. Among various synthetic substrate analogs, both enantiomers of CDP-unsaturated archaeols with ether-linked geranylgeranyl chains and CDP-saturated archaeol with ether-linked phytanyl chains were similarly active toward the archaetidylserine synthase. The activity on the ester analog of the substrate was two to three times higher than that on the corresponding ether-type substrate. The activity of D-serine with the enzyme was 30% of that observed for L-serine. A trace amount of an acid-labile, unsaturated archaetidylserine intermediate was detected in the cells by a pulse-labeling experiment. A gene (MT1027) in M. thermautotrophicus genome annotated as the gene encoding phosphatidylserine synthase was found to be homologous to Bacillus subtilis pssA but not to Escherichia coli pssA. The substrate specificity of phosphatidylserine synthase from B. subtilis was quite similar to that observed for the M. thermautotrophicus archaetidylserine synthase, while the E. coli enzyme had a strong preference for CDP-1,2-diacyl-sn-glycerol. It was concluded that M. thermautotrophicus archaetidylserine synthase belongs to subclass II phosphatidylserine synthase (B. subtilis type) on the basis of not only homology but also substrate specificity and some enzymatic properties. The possibility that a gene encoding the subclass II phosphatidylserine synthase might be transferred from a bacterium to an ancestor of methanogens is discussed.

    INTRODUCTIONhg1)h, 百拇医药

    Major polar lipids of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum [27]) {Delta} H have been reported to be diether- and tetraether-type phospholipids, glycolipids, and phosphoglycolipids containing L-serine, ethanolamine, myo-inositol, and ß-D-glucosyl-(1-6)-ß-D-glucose as polar head groups. Considering these polar lipid structures and other known structures of archaeal phospholipids, three common and specific characteristics of archaeal polar lipids are recognized. First, all the polar lipids consist of ether linkages between glycerophosphate (GP) and hydrocarbon chains; second, the hydrocarbon chains are exclusively isoprenoid alcohols; and third, the most exclusive feature of archaeal phospholipids is the stereoconfiguration of the GP backbone. The GP backbone of phospholipid in the Archaea is sn-glycerol-1-phosphate (G-1-P), which is the enantiomer of its bacterial and eucaryotic counterparts (10). The mechanism by which these specific characteristics are formed is now partly elucidated through in vitro studies of polar lipid biosynthesis in Archaea, which have already revealed four enzymatic reactions and their substrate specificities (14, 16, 28, 29) Dihydroxyacetone phosphate, which is an intermediate of glycolysis and gluconeogenesis, is the starting substrate of polar lipid biosynthesis . Dihydroxyacetone phosphate is first reduced exclusively to G-1-P by G-1-P dehydrogenase (Fig. 1, reaction 1). The activity of G-1-P dehydrogenase or the genes encoding G-1-P dehydrogenase have been detected in all archaeal species studied so far (18). In the next two steps, G-1-P is bound to isoprenoid hydrocarbons through two ether bonds at the sn-2 and -3 positions of the glycerol moiety (28, 29) ( reactions 2 and 3). The ether bond-forming enzymes are specific to G-1-P and geranylgeranyl pyrophosphate, so the product of these steps is 2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate (unsaturated archaetidic acid). Genes encoding the first ether bond-forming enzyme are detected in seven species of archaeal genomes (23). The intermediate formed is then activated by CTP to form CDP-unsaturated archaeol by the action of CTP:2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate cytidyltransferase (14) ( reaction 4). All these studies on polar lipid biosynthesis in Archaea were carried out for M. thermautotrophicus and its closest relative, Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum strain Marburg [27]), and this pathway is analogous to the bacterial pathway of phospholipid biosynthesis except for the structural differences in stereostructure of GP, ether bonds, and hydrocarbon chains. CDP-diacylglycerol, which corresponds to CDP-unsaturated archaeol, plays a central role in the biosynthesis of a number of phospholipids in Bacteria (4). In order to prove the next reaction of polar lipid biosynthesis in Archaea ( reaction 5), we began to look for an enzyme capable of synthesizing serine phospholipid in M. thermautotrophicus using the well-known bacterial pathway. We found such an activity (14), which catalyzed the formation of archaetidylserine from CDP-archaeol and L-serine (archaetidylserine synthase, e.g., CDP-2,3-di-O-geranylgeranyl-sn-glycerol: L-serine O-archaetidyltransferase) according to the reaction 5 shown in .

    fig.ommitted;d, 百拇医药

    Enzymatically identified reactions of biosynthesis of polar lipids in Archaea. Reactions 1 to 4 have already been reported (14, 16, 28, 29). Reaction 5 is described in the present work. DHAP, dihydroxyacetone phosphate; PP, pyrophosphate.;d, 百拇医药

    Studying archaeal polar lipid biosynthesis, in particular the step of the attachment of the polar head group, one may expect to encounter several questions concerning the specific archaeal lipid structures. One of the questions is how archaetidylserine synthase contributes to the establishment and selection of the specific features of archaeal polar lipid structure. The enzymes that catalyze reactions 1 to 3 in are specific for G-1-P and its derivatives of the same stereoconfiguration. By contrast, CDP-archaeol synthase, which catalyzes reaction 4, does not recognize the structure of the GP backbone or the ether or ester bonds between GP and hydrocarbon chains but mainly targets a substrate possessing geranylgeranyl chains. The second question deals with the relationship between archaetidylserine synthase and bacterial or eukaryal phosphatidylserine synthases. Because homologies of the gene encoding phosphatidylserine synthase (pssA) in Bacillus subtilis, but not in Escherichia coli, were found in several archaeal species whose whole genome sequences are known, the enzymatic properties which cannot be inferred from their genome sequences should be compared with those of the analogous enzymes. This might give a clue to the origin of amino group-containing phospholipids in Archaea. The other problem is the exact sequence of polar group attachment and hydrogenation of unsaturated hydrocarbon chains, which contrasts with completely saturated chains in the final products.

    The present work reports some properties and the substrate specificity of archaetidylserine synthase in M. thermautotrophicus and compares them to those of bacterial phosphatidylserine synthase. The nomenclature of archaeal lipids proposed by Nishihara and Koga (15) is used throughout this paper.he6s, 百拇医药

    MATERIALS AND METHODShe6s, 百拇医药

    Growth of microorganisms. M. thermautotrophicus {Delta} H (DSM 1053) and E. coli DSM 1649 were grown as previously described (14). B. subtilis JCM 1465 was grown with shaking at 37°C for 6 h in a 3-liter Erlenmeyer flask in 800 ml of JCM medium 22 (Japan Collection of Microorganisms catalogue of strains, 1999) supplemented with 1% glucose.he6s, 百拇医药

    Chemical synthesis of CDP-archaeol and CDP-diacylglycerol. CDP-2,3-di-O-geranylgeranyl-sn-glycerol and CDP-2,3-di-O-phytanyl-sn-glycerol were chemically synthesized with cytidine 5'-monophosphomorpholidate from corresponding archaetidic acid as previously described (9, 14). Other substrates, CDP-1,2-di-O-geranylgeranyl-sn-glycerol and CDP-1,2-di-O-oleyl-sn-glycerol (diether type) and CDP-diacylglycerols (diester type, CDP-2,3-di-O-oleoyl-sn-glycerol and CDP-1,2-di-O-acyl-sn-glycerol) were synthesized as previously described (9, 14). L-{alpha} -phosphatidic acid from egg yolk lecithin (Nacalai, Kyoto, Japan) was used as a starting material for the synthesis of CDP-1,2-di-O-acyl-sn-glycerol.

    Enzymatic preparation of [3H]CDP-unsaturated archaeol. [Cytosine-5-3H]CDP-unsaturated archaeol was enzymatically synthesized as described in the previous paper (14) except that 15 µCi of [5-3H]CTP (0.625 Ci/mol) was used in 1.2 ml of the reaction mixture. After incubation for 5 h, a chloroform-soluble fraction was obtained from the reaction mixture and then [cytosine-5-3H]CDP-unsaturated archaeol was purified by acidic-alkaline partitioning (14).(:, 百拇医药

    TLC. Thin-layer chromatography (TLC) gel was developed on a Silica Gel 60 plate (Merck) with the following solvents: solvent A, chloroform-methanol-7 M ammonia (60:35:8); solvent B, chloroform-methanol-acetic acid-water (80:30:15:5). Spots of amino group-containing lipids were visualized by spraying with ninhydrin reagent. Authentic archaetidylserine was isolated from M. thermautotrophicus cells as previously described (17). Water-soluble products were developed on a thin-layer cellulose plate (Merck 5716) with phenol-water (100:38). Standard CMP was purchased from Kohjin, Tokyo, Japan. Radioactive spots were recorded by a Fujifilm Fluor image analyzer (model FLA-2000) with an imaging plate: Fujifilm type BAS-TR for 3H-labeled lipids and type BAS-MS for 32P- and 33P-labeled lipids.

    Preparation of cell-free homogenates. Frozen M. thermautotrophicus cells (about 20 g wet weight) suspended in 25 ml of 10 mM Bicine buffer (pH 8.0) containing 1 mM MgCl2, 5 mM 2-mercaptoethanol (buffer M), and 1 mg of DNase I (Sigma) were passed through a French pressure cell operated at 1,400 kg/cm2. This process was repeated twice. Cell debris and unbroken cells were removed by centrifugation (10,000 x g) for 10 min. The homogenate was stored at -20°C until further use. The membrane fraction was obtained by centrifugation at 100,000 x g for 2 h. The pelleted membrane fraction was resuspended in buffer M. Crude cell extracts of B. subtilis and E. coli were prepared as previously described (5, 6).l, 百拇医药

    Enzyme assay. The complete assay mixture (final volume, 0.2 ml) for archaetidylserine synthase of M. thermautotrophicus contained 0.5 mM CDP-archaeol, 10 mM [3-3H]L-serine (1.25 Ci/mol; Amersham Pharmacia Biotech), the cell-free homogenate of M. thermautotrophicus (568 µg of protein), 0.125 M Bicine buffer (pH 8.5), l% Triton X-100, and 10 mM MnCl2. After incubation at 60°C for 10 min, the reaction was stopped by the addition of 1 ml of 0.1 M HCl in methanol, 2 ml of chloroform, and 3 ml of 1 M MgCl2. Chloroform-extractable 3H material was separated from water-soluble components by phase partitioning, and radioactivity was counted. In the case of determination of stereospecificity to serine of archaetidylserine synthase, [3-3H]L-serine was replaced by nonradioactive D- or L-serine in 0.8 ml of the reaction mixture. After incubation at 60°C for 30 min, phospholipid was precipitated by the addition of acetone to a chloroform-soluble fraction in order to remove Triton X-100 (14). The precipitate of phospholipids was developed on TLC with solvent B. A spot corresponding to archaetidylserine was visualized by spraying with acid molybdate reagent, and the phosphorus content of the spot scraped off from the plate was determined (1). The activity of archaetidylserine synthase was calculated based on the formation of archaetidylserine over 30 min measured by phosphate determination. The phosphorus content of the spot corresponding to archaetidylserine in a control experiment without D- or L-serine was subtracted from the total phosphorus of archaetidylserine determined for the TLC spot.

    Phosphatidylserine synthase activity was measured in B. subtilis and E. coli as previously described (5, 6). Protein content was determined by the bicinchoninic acid method (22).mg, 百拇医药

    Identification of the reaction product. To obtain a large amount of the enzyme reaction product for structural analysis, 20 times more reactants were incubated for 30 min. Nonradioactive L-serine replaced L-[3H]serine. The product was extracted and purified by acetone precipitation and TLC as described above. The fast atom bombardment-mass spectrum of the product was recorded with a mass spectrometer (JEOL JMS DX-303) with a matrix of m-nitrobenzyl alcohol containing a small amount of NaI in a positive mode. The presence of an allyl ether linkage was checked by the lability to the treatment of 5% HCl-methanol at 80°C for 1 h. For the identification of water-soluble product, [cytosine-5-3H]CDP-archaeol (290,000 dpm) was reacted with unlabeled L-serine in a standard reaction mixture. The water-soluble radioactive product was recovered in the aqueous phase after Bligh-Dyer partitioning and was developed by cellulose-TLC.

    Detection of allyl ether containing archaetidylserine in M. thermautotrophicus cells. M. thermautotrophicus was grown successively twice in 50 ml of low-phosphate medium (17) containing 100 µCi of 33Pi (5 Ci/mol) under a pressurized atmosphere of H2 + CO2 + H2S (78:22:0.2) in a 500-ml flask with shaking for 24 h. Finally, 50 ml of the same medium containing the same specific radioactivity of 33Pi was inoculated with 5 ml of the last subculture. At the early logarithmic phase of growth (after incubation for 7 h), 2 mCi of 32Pi was added and the culture was allowed to continue to grow. After 10 min, cells were harvested and total lipid was extracted. The radioactive phospholipid corresponding to archaetidylserine was purified from the total lipid by two-dimensional TLC. The isolated archaetidylserine was treated with 5% HCl-methanol at 80°C for 1 h to degrade allyl ether archaetidylserine. The degradation products were partitioned into chloroform-soluble and water-soluble fractions. 32P and 33P radioactivities in the chloroform-soluble products, aqueous products, and the untreated archaetidylserine were counted using a liquid scintillation analyzer (Packard TRI-CARB 2700TR) with Aquasol-2 (Packard) as a scintillator.

    RESULTS&+unv5, http://www.100md.com

    Identification of the reaction product. A cell-free homogenate of M. thermautotrophicus catalyzed the conversion of [3-3H]serine into chloroform-extractable 3H-labeled material in the presence of CDP-2,3-di-O-geranylgeranyl-sn-glycerol. The chloroform-soluble 3H-labeled product of the archaetidylserine synthase reaction showed a single spot comigrating with authentic archaetidylserine when chromatographed on a TLC plate with solvent A (Rf = 0.33) and solvent B (Rf = 0.43). When several kinds of substrate analogs, that is, CDP-1,2-di-O-geranylgeranyl-sn-glycerol, CDP-2,3-di-O-phytanyl-sn-glycerol, and CDP-rac-di-O-oleoyl-glycerol were used instead of CDP-2,3-di-O-geranylgeranyl-sn-glycerol, identical results were obtained. While it might be expected that archaetidylethanolamine is formed from archaetidylserine by a decarboxylation reaction, as suggested by an in vivo pulse-chase experiment (17), no spot corresponding to archaetidylethanolamine was detected on the TLC plate after the archaetidylserine synthase reaction.

    The reaction product from CDP-2,3-di-O-geranylgeranyl-sn-glycerol was also chemically and mass-spectrometrically analyzed. The lipid product enzymatically prepared from CDP-2,3-di-O-geranylgeranyl-sn-glycerol was purified by TLC with solvent B. The fast atom bombardment-mass spectrum of the lipid product gave signals of m/z 803 (M)+, m/z 827 (M + Na + H)+, m/z 762 (M - serine + 2Na+H)+, and m/z 784 (M - serine + 3Na)+, which were consistent with the structure of archaetidylserine with geranylgeranyl groups as hydrocarbon chains. The presence of allylic ether linkages was also suggested by the acid lability. The product of the enzyme reaction was completely degraded by treatment with 5% HCl-methanol at 80°C for 1 h. These results suggest that the product from CDP-unsaturated archaeol most likely is 2,3-di-O-geranylgeranyl-sn-glycero-1-phosphoserine (unsaturated archaetidylserine), even though the individual components and the stereostructure of the product were not completely determined. We also analyzed the water-soluble product of the reaction with [cytosine-5-3H]CDP-archaeol and unlabeled L-serine as substrates. One radioactive spot (Rf = 0.33) was found that cochromatographed with standard CMP on a cellulose TLC plate.

    A nonradioactive by-product was detected on the TLC with an Rf of 0.19. The compound was positively stained with acid molybdate reagent on the TLC plate. For an analogous reaction, Walton and Goldfine (26) reported that phosphatidyltransferase from Clostridium butyricum catalyzed the transfer of the phosphatidyl moiety of phospholipid to Triton X-100, and in vitro formation of phosphatidyltriton was observed. Therefore, it was assumed to be a Triton X-100 adduct of an archaetidyl group (e.g., archaetidyltriton X-100) and was not further analyzed.@1y$', http://www.100md.com

    Properties of archaetidylserine synthase. Under the assay conditions used, radioactivity was incorporated into a chloroform-soluble fraction almost linearly for 30 min, and then the rate gradually slowed. Approximately 30 nmol of archaetidylserine was formed when 100 nmol of CDP-archaeol was incubated in the reaction mixture for 1 h . In routine assays, the incubation time was 10 min. Archaetidylserine synthase activity was roughly linear with protein concentration under the assay conditions (data not shown). The effect of nonionic detergent Triton X-100 on archaetidylserine synthase activity is shown in . Triton X-100 was required for archaetidylserine synthase activity and maximum activity was obtained at a concentration of 1%. The enzyme activity was stimulated by the addition of Mn2+, with maximum activities occurring at concentrations of 5 mM or more. The addition of Mg2+ had much less effect on the enzyme activity . These results show that the enzyme activity was dependent on addition of Triton X-100 and Mn2+ ion. The enzyme did not require the addition of K+ ion. The enzyme activity was lowered to about 70% of its maximum at 0.6 M K+, which corresponds to the intracellular concentration found in M. thermautotrophicus (20) . Maximal enzyme activity was observed at pH 8.0 to 8.5 (Bicine buffer) (data not shown) and at 60°C . The membrane fraction and cell supernatant, respectively, contained 61 and 32% of the total activity found in the cell-free homogenate of M. thermautotrophicus. Specific activities of cell-free homogenates, membrane fraction and cell supernatant were 64, 146, and 19 nmol/h/mg of protein, respectively. Unfractionated cell-free homogenate was usually used for further studies.

    fig.ommittedwh|ed), 百拇医药

    onversion of [3H]serine to lipid catalyzed by M. thermautotrophicus homogenate incubated with CDP-2,3-di-O-geranylgeranyl glycerol in the presence of 10 mM MnCl2 and 1% Triton X-100 at 60°C, pH 8.5. AS, archaetidylserine.wh|ed), 百拇医药

    fig.ommittedwh|ed), 百拇医药

    Effects of Triton X-100 concentration (A), Mn2+ and Mg2+ concentration (B), K+ concentration (C), and temperature (D) on archaetidylserine (AS) synthase activity. The conditions of the experiments were the same as described in Materials and Methods with CDP-2,3-di-O-geranylgeranyl glycerol except that the K+ concentration was 0.5 M for panels A and B, and the reaction was performed at 55°C for panels A, B, and C.wh|ed), 百拇医药

    Substrate specificity of archaetidylserine synthase. One of the major questions concerning archaeal lipid biosynthesis is how and at which step the specific structural characteristics of archaeal lipids are formed. The substrate stereospecificity of archaetidylserine synthase was, therefore, examined using a variety of chemically synthesized substrate analogs listed in . The list includes CDP-archaeol (substrates 1 to 4)/CDP-diacylglycerol (substrates 5 and 6) analogs with both stereoisomers of the GP backbone, ether and ester bonds between GP and hydrocarbons, unsaturated and saturated isoprenoid hydrocarbon chains, and straight-chain hydrocarbons. Archaetidylserine synthase of M. thermautotrophicus showed similar activities when either enantiomer of CDP-archaeol possessing geranylgeranyl chains (substrates 1 and 2) was used as a substrate. The substrate analogs with saturated ones (substrate 3) or straight hydrocarbon chains (substrate 4) showed slightly lower activities, but the activity range was between less than ±50% of the activity on the natural substrate (substrate 1). Interestingly, when ester-lipids containing straight aliphatic chains (substrates 5 and 6) were used, activities were two to three times higher than those observed with an ether-type substrate (substrate 1).

    fig.ommittedw1w), 百拇医药

    Substrate specificities of archaetidylserine synthase and phosphatidylserine synthasew1w), 百拇医药

    The stereospecificity of archaetidylserine to L- or D-serine was determined using nonradioactive substrates. Archaetidylserine synthase preferred L-serine to D-serine. The relative activity for D-serine was 32% ± 6% (n = 2) of what was observed with L-serine.w1w), 百拇医药

    Substrate specificity of bacterial phosphatidylserine synthase. Because archaetidylserine synthase is known to be homologous to phosphatidylserine synthase from B. subtilis (12), the substrate specificities of phosphatidylserine synthase from B. subtilis and another type of phosphatidylserine synthase from E. coli were compared to that of archaetidylserine synthase. B. subtilis phosphatidylserine synthase showed almost similar activities in every case when the substrates listed in Table 1 were used. That is, the enzyme did not discriminate between the stereostructures of the GP backbone, ether or ester linkage, and hydrocarbon chains of the analogs of CDP-archaeol. The substrate specificity of the B. subtilis enzyme was quite similar to that of the methanogen's archaetidylserine synthase described above. By contrast, E. coli phosphatidylserine synthase appeared to distinguish between such differences in the substrate structures. A drastic decrease in activity was observed when the mirror image isomer (CDP-2,3-diacyl-sn-glycerol [, substrate 5]) of the natural substrate (substrate 6) or the ether analogs with isoprenoid chain (substrates 1 to 3) were incubated with E. coli cell extracts. An ether-type substrate with natural GP stereostructure and straight-chain hydrocarbons (CDP-1,2-dioleyl-sn-glycerol [substrate 4]) revealed low but significant activity (41%) when compared with the ester-type natural substrate (substrate 6). In other words, substitution of ester linkages in the substrate structure showed a significant effect on the activity of E. coli phosphatidylserine synthase. Thus, a difference between phosphatidylserine synthases from both bacterial species was demonstrated also in substrate specificity.

    Intracellular occurrence of allyl ether-type archaetidylserine in M. thermautotrophicus cells. In order to elucidate whether unsaturated (or allyl ether-type) archaetidylserine is really formed in the cells, we tried to detect it on the basis of the acid lability of allyl ether lipids. An allyl ether bond is degraded with 5% HCl-methanol at 80°C for 1 h (14). On the other hand, saturated archaetidylserine is stable to acid treatment because nonallyl ether bonds are not readily hydrolyzed and the phosphodiester bond cannot be hydrolyzed by cyclic phosphodiester formation due to the lack of a free hydroxyl group on serine residues (11). M. thermautotrophicus was continuously labeled with 33Pi and pulse-labeled with 32Pi for 10 min in the presence of 33Pi. In this experiment, 33P in archaetidylserine must represent the amount of mature archaetidylserine (the final product of the biosynthetic pathway) with saturated hydrocarbons, while 32P must express the amount of newly synthesized archaetidylserine. Labeled archaetidylserine was purified from the total lipid of the cells. The purified archaetidylserine was decomposed with 5% HCl-methanol at 80°C for 1 h. The ratio of radioactivities of 32P versus 33P (32P/33P) of chloroform-soluble products and water-soluble products after acid treatment of archaetidylserine was compared to that of untreated archaetidylserine. The 32P/33P ratio (1.26) of untreated archaetidylserine decreased by 17% to 1.04 in chloroform-soluble products (acid-stable lipids) after acid treatment. On the other hand, the ratio (2.17) in the aqueous fraction after acid treatment (acid-labile degradation products) was 1.7 times higher than the ratio (1.26) observed in untreated archaetidylserine. These results suggest that only a trace amount of newly synthesized allyl ether-type archaetidylserine is present in the cells. Almost all archaetidylserines have fully saturated hydrocarbon chains, as shown by chemical analysis (17).

    DISCUSSIONe, 百拇医药

    The results described above confirm the presence of archaetidylserine synthase in M. thermautotrophicus homogenate. The enzyme catalyzed the transfer of an archaetidyl group from CDP-archaeol to serine. When CDP-unsaturated archaeol was used as a substrate, the product was identified as archaetidylserine with geranylgeranyl chains. The water-soluble product of the reaction was identified as CMP. The stoichiometry could not be established because Triton X-100 interfered with the quantitative recovery of lipids from the reaction mixture and because of the formation of by-products such as archaetidyltriton X-100. The amount of lipid (30 nmol) synthesized during the enzyme reaction for 1 h was much more than the amount of endogenous allyl ether-type phospholipid (at most 0.17 nmol, see reference 14) in the enzyme source (the cell-free homogenate, 568 µg of protein, containing 36 nmol of total phospholipid [15]), excluding the possibility that the putative substrate might simply stimulate incorporation of radioactive serine into an endogenous allyl ether-type lipid.

    Archaetidylserine synthase showed a loose specificity for CDP-archaeol analogs; that is, the enzyme is able to utilize CDP-archaeol analogs with both enantiomers of the GP backbone, ester and ether linkages, and unsaturated and saturated isoprenoid and straight-chain fatty acid. This means that archaetidylserine synthase is not involved in establishing the specific features of archaeal polar lipid structures. According to knowledge obtained so far, the first three enzymes in the biosynthesis pathway of the archaeal polar lipid appear to play a central role in the formation of the specific features of archaeal polar lipid structures, while the ensuing steps do not. Archaetidylserine synthase preferred L-serine over D-serine. Serine stereospecificity of phosphatidylserine synthase in Bacteria has not been reported.a|, http://www.100md.com

    There are two genetically distinct subclasses of phosphatidylserine synthase. The enzymes classified in subclass I are distributed in gram-negative bacteria (e.g., E. coli). The subclass II enzymes have widespread distribution in gram-positive bacteria (e.g., B. subtilis), yeast, and Archaea (Methanococcus jannaschii) (12). It is known that there are some different properties between E. coli-type phosphatidylserine synthase (subclass I) and B. subtilis phosphatidylserine synthase (subclass II). For example, their intracellular localization, divalent cation requirement, and reaction mechanisms are different (7). In addition, the enzymes from E. coli and B. subtilis show no sequence homology (19). An M. jannaschii gene encoding archaetidylserine synthase is known to belong to subclass II (12). A gene (MT1027) homologous to the B. subtilis phosphatidylserine synthase gene (pssA) has been identified in the M. thermautotrophicus genome (21). The gene MT1027 was annotated as phosphatidylserine synthase. The amino acid sequence data of phosphatidylserine synthase of B. subtilis (P39823), M. thermautotrophicus (A69004 = MT1027), and Methanocaldococcus jannaschii (Q58609 = MJ1212) were obtained at the NCBI site . A multiple alignment of the three sequences was constructed with an alignment software, CLUSTAL W 1.7 . Although the archaetidylserine synthase characterized in this work has not been cloned and sequenced, it is most likely that MT1027 codes for archaetidylserine synthase. The deduced amino acid sequence of MT1027 showed significant homology with B. subtilis phosphatidylserine synthase (23.1% identity and 52.6% similarity), whereas MT1027 showed no homology with that of the E. coli enzyme. In addition to finding homology in the primary structures, this work also demonstrated similarities in some biochemical properties (requirements of Triton X-100 and Mn2+ ion) of archaeal and Bacillus enzymes. Substrate specificity is the other characteristic used for comparison of archaeal archaetidylserine synthase with subclass I and II phosphatidylserine synthases. We investigated lipid substrate specificity of archaetidylserine synthase from M. thermautotrophicus and phosphatidylserine synthases from B. subtilis and E. coli. The data described in this paper confirm the results presented by Carman and Dowhan on the lipid substrate stereoisomer specificity of phosphatidylserine synthase from E. coli (3): they showed that the purified enzyme was specific for the sn-glycero-3-phosphate (G-3-P) isomer of the liponucleotide and did not recognize the G-1-P isomer in a kinetic study using CDP-1,2-dipalmitoyl-sn-glycerol and CDP-1,2-dipalmitoyl-rac-glycerol as substrates. In addition, our work showed that E. coli phosphatidylserine synthase recognized ester bonds between GP and the hydrocarbon chains and nonbranched hydrocarbon chain. On the other hand, B. subtilis phosphatidylserine synthase revealed loose substrate specificity like M. thermautotrophicus archaetidylserine synthase. Therefore, we conclude that archaeal archaetidylserine synthase is a member of the Bacillus-type phosphatidylserine synthase family (subclass II) not only on the basis of amino acid sequence homology but also from the enzymatic properties including substrate specificity.

    fig.ommittedg;22, http://www.100md.com

    Multiple alignment of phosphatidylserine synthase and relatives. Abbreviations: Mja, M. jannaschii (201 amino acids); Mth, M. thermautotrophicus (233 amino acids); Bsu, B. subtilis (177 amino acids).g;22, http://www.100md.com

    It may be inferred from our results and other reports that archaeal archaetidylserine synthase and Bacillus phosphatidylserine synthase originated from a common ancestral enzyme. Because amino group-containing phospholipids are confined only to methanogens and some related Euryarchaeota among the Archaea (11, 24) and widely distributed in bacteria (8), it is speculated that the gene encoding the ancestral enzyme was transferred from a gram-positive bacterium possessing subclass II phosphatidylserine synthase to a group including methanogens after differentiation from other Archaea groups. A symbiotic relationship between methanogens and some kind of hydrolytic fermentative bacteria via interspecies hydrogen transfer in anaerobic environments (2, 25) would give an increasing chance to exchange genes. This speculation is supported by the fact that the archaetidylserine synthase activities of ester lipids are twice (or more) those of ether lipids. Bacillus phosphatidylserine synthase can catalyze the formation of archaetidylserine from CDP-archaeol and L-serine (, substrate 1). It could be also speculated that, when the pssA gene was transferred, the loose substrate specificity of the subclass II phosphatidylserine synthase would be the most adequate to catalyze reaction 5 in the biosynthesis of the enantiomeric ether phospholipid in an archaeon, if substrate specificity at that time was the same as at present. The subclass I (E. coli-type) phosphatidylserine synthase could not do this task because of the strict specificity. Although the intracellular salt (K+) concentration (0.6 M) in M. thermautotrophicus is not optimal for activity, the enzyme is still active at the intracellular K+ concentration. This is in contrast to CDP-archaeol synthase, which shows no homology with bacterial enzymes and exhibits maximum activity in the presence of 0.5 M K+ (14). This fact is consistent with the speculation that the gene encoding archaetidylserine synthase was transferred from Bacteria to Archaea.

    Although the three preceding enzymes involved in the ether bond formation (, reactions 2 and 3) and the activation of the intermediate by CDP of phospholipid biosynthesis ( reaction 4) have been shown to be specific to geranylgeranyl chains, archaetidylserine synthase (, reaction 5) has not. Archaetidylserine synthase reacted with substrates with both saturated and unsaturated isoprenoid chains. The cellular polar lipids have fully saturated hydrocarbon chains. Therefore, there should be steps of hydrogenation (saturation) of geranylgeranyl chains somewhere before (reaction 3 in) or after (reaction 2 in Fig. 5) the step of archaetidylserine formation. The exact sequence is not known. In order to obtain a clue to clarify this problem, the presence of an unsaturated archaetidylserine intermediate was surveyed based on the acid lability of geranylgeranyl ethers. An in vivo pulse-label experiment with 32Pi revealed the intracellular presence of newly synthesized acid-labile (probably allyl ether-bonded) archaetidylserine. This result is consistent with the work of Moldoveanu and Kates (13), in which they demonstrated the presence of acid-labile unsaturated ether intermediates of phospholipid biosynthesis in the extremely halophilic archaeon Halobacterium cutirubrum by pulse-labeling and chase experiments. The detection of an acid-labile archaetidylserine intermediate indicates that at least an allyl ether-bonded archaetidylserine intermediate is really present in the cells which are involved in phospholipid biosynthesis. The presence of an allyl ether serine-containing intermediate suggests that the biosynthetic pathway via reactions 1 and 2 in is probably operated in the cells, although the possibility of another pathway via reactions 3 and 4 is not excluded because archaetidylserine synthase did react with saturated CDP-archaeol and the intracellular absence of saturated CDP-archaeol has not been excluded.

    fig.ommitted4, 百拇医药

    Possible biosynthetic pathways of archaetidylserine from unsaturated archaetidic acid. CDP-archaeol synthase is known to be specific to unsaturated archaetidic acid (14). Because archaetidylserine synthase can react with both saturated and unsaturated CDP-archaeol, two pathways (reactions 1 + 2 and reactions 3 + 4) to form saturated archaetidylserine are possible. *, allyl ether bond.4, 百拇医药

    ACKNOWLEDGMENTS4, 百拇医药

    We are greatly indebted to M. Murakami and N. Asakawa (Eisai Co., Ltd.) for supplying us with geranylgeraniol. We also thank M. Ohga for help in growing microorganisms in fermentors.4, 百拇医药

    This work was partly supported by Grant-in-Aid for Scientific Research B 11460051 from the Ministry of Education, Science, Sports, and Culture of Japan.4, 百拇医药

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